1. Field of the Invention
The present invention relates to a method for diagnosis of mycobacterial infection by comparative sequence analysis of rpoB gene coding for .beta.-subunit of RNA polymerase, more specifically, to a method for detecting and identifying mycobacterial species which comprises steps of amplifying 342 bp of rpoB gene fragments from clinically isolated mycobacteria using mycobacterial rpoB-specific PCR primers; sequencing 306 bp regions of the amplified 342 bp of rpoB gene fragments except the primer region; and, inferring a phylogenetic tree with reference species.
2. Description of the Prior Art
The genus Mycobacterium covers a wide range of organisms including obligate parasites causing serious human and animal diseases such as tuberculosis, bovine tuberculosis and leprosy, opportunistic pathogens, and saprophytic species found in natural environment (see: Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, Manual of Clinical Microbiology, 6th Ed. ASM Press, Washington, D.C., Frederick S. N., B. Metchock, pp. 400-437(1995)).
Recently, in line with rapid increase of AIDS patients worldwide, infections with nontuberculous mycobacteria or mycobacteria other than Mycobacterium tuberculosis (hereinafter, referred to as "MOTT") or Mycobacterium leprae go on increasing (see: Barnes, P., A. B. Bloch, P. T. Davidson and D. E. Snider, Jr., Tuberculosis in Patients with Immunodeficiency Virus Infection, New Engl. J. Med., 324:1644-1650(1991)).
In general, mycobacteria have been largely classified into four groups depending on growth rate and pigmentation of colonies(see: Runyon, E. H., Identification of Mycobacterial Pathogens Utilizing Colony Characteristics, Am. J. Clin. Pathol., 54:578-586(1970)), and numerical taxonomic analysis(see: Sneath, P. H. A. and R. R. Sokal, Numerical Taxonomy, W.H. Freeman & Co., San Francisco (1973)), immunological techniques (see: Wayne, L. G., R. C. Good, A. Tsang, R. Buttler, D. Dawson, D. Groothuis, W. Gross, J. Hawkins, J. Kilburn, M. Kubin, K. H. Schroder, V. A. Silcox, M.-F. Thorel, C. Woodley and M. A. Yakrus, Serovar Determination and Molecular Taxonomic Correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: A Cooperative Study of the international Working Group on Mycobacterial Taxonomy, Int. J. Syst. Bacteriol., 43(3):482-489(1993)), comparison of cell wall composition, DNA-DNA homology, and analysis employing restriction endonucleases (see: Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Botter and T. Bodmer, Rapid Identification of Mycobacteria to the Species Level by Polymerase Chain Reaction and Restriction Enzyme Analysis, J. Clin. Microbiol., 31(2):175-178(1993)) have been used to classify mycobacterial species more definitely.
However, the conventional methods for identifying mycobacterial species have revealed disadvantages that they are laborious and complex, and require so long time. Naturally, alternative methods for identification employing a gene as a marker, e.g., genus-specific or species-specific PCR primers or nucleic acid probe against a specific gene have been used in the art.
Under the circumstances, mycobacterial phylogenetic analysis to provide a criterion of differentiation and identification of mycobacterial species has been performed based on the sequences of 16S rRNA or its coding gene (16S rDNA), which provided a fact that 16S rRNA-based phylogenetic analysis permits to define mycobacterial phylogenetic relationships well (see: Stahl, D. A. and J. W. Urbance, The Division between Fast and Slow-growing Species Corresponds to Natural Relationships among the Mycobacteria, J. Bacteriol., 172:116-124(1990); Rogall, T., T. Flohr and E. C. Bottger, Differentiation of Mycobacterium Species by Direct Sequencing of Amplified DNA, J. Gen. Microbiol., 136(Pt9):1915-1920(1990); Rogall, T., J. Wolters, T. Flohr and E. C. Bottger, Towards a Phylogeny and Definition of Species at the Molecular Level within the Genus Mycobacterium, Int. J. Syst. Bacteriol., 40:323-330(1990)).
However, 16S rRNA-based phylogenetic analysis has a shortcoming that clear definition of species boundaries is often difficult (for example, in the case of slow-growing mycobacteria) (see: Fox, G. E., J. D. Wisotzkey and P. J. Jurtshumk, How Close IS Close: 16S rRNA Sequence Identitiy May Not Be Sufficeint to Guarantee Species Identity, Int. J. Syst. Bacteriol., 42:166-170(1992)).
Thus, a dnaJ gene coding for a stress protein has been suggested as a promising alternative (see: Takewaki, S. K. Okuzumi, H. Ishiko, K. Nakahara, A. Ohkubo and R. Nagai, Genus-specific Polymerase Chain Reaction for the Mycobacterial dnaj Gene and Species-specific Oligonucleotide Probes, J. Clin. Microbiol., 31:446-450(1993); Takewaki, S., K. Okuzumi, I. Manabe, M. Tanimura, K. Miyamura, K. Nakahara, Y. Yazaki, A. Ohkubo and R. Nagai, Nucleotide Sequence Comparison of the Mycobacterial dnaJ Gene and PCR-restriction Fragment Length Polymorphism Analysis for Identification of Mycobacterial Species, Int. J. Syst. Bacteriol., 44:159-166(1994)). However, it was found that dnaJ-based phylogenetic analysis has several problems unsuitable for differentiation of rapid-growing mycobacteria.
The said nucleic acid probes to employ 16S rRNA gene as a marker are clear criteria for defining 5 kinds of mycobacterial species, e.g., M. tuberculosis, M. bovis, MAC, M. kansasii and M. gordonae, and they are commercially available in the art (AccuProbe: Gen-Probe, San Diego, Calif., USA) (see: Nolte, F. S. and Beverly Metchock, Ch. 31. Mycobacterium in Manual of Clinical Microbiology, pp. 400-437(1995)).
Also, IS6110 insertion element which exists in TB complex (M. tuberculosis, M. africanum and M. bovis) in multiple copies, has been employed as a marker in a PCR detection method. However, the result obtained through the said method maybe false negative, since Mycobacterium tuberculosis free of the insertion element has been reported (see: Yuen, L. K., B. C. Ross, K. M. Jackson and B. Dwyer, Characterization of Mycobacterium tuberculosis Strains from Vietnamese Patients by Southern Blot Hybridization, J. Clin. Microbiol., 31:1615-1618(1993)). Primers to amplify the said gene are commercially available now (TB-CR, TB Detection kit, Bioneer Co., Korea), though they play a limited role of detecting mycobacterial species, and can not practically be applied in identifying mycobacteria as well as detecting existence of mycobacteria.
When the afore-mentioned 5 kinds of probes are used, typical mycobacterial species causing human diseases can be identified. However, the probes have a shortcoming of cross-hybridization with newly described species (see: Buttler, W. R., S. P. O'connor, M. A. Yakrus and W. M. Gross, Cross-reactivity of Genetic Probe for Detection of Mycobacterium tuberculosis with Newly Described Species Mycobacterium celatum, J. Clin. Microbiol., 32(2):536-538(1994)). Also, infections with MOTT other than Mycobacterium tuberculosis increase, MAIS (Mycobacterium avium-intracellulare-scrofulaceum) complex among MOTT infects human frequently, and infections with new species have been reported continuously. Accordingly, there are strong reasons for clear definition of species causing diseases to prevent and control the diseases, and development of a novel nucleic acid probe showing species-specific genetic difference is strongly required in the art.
Under the circumstances, the present inventors have investigated whether a rpoB gene coding for .beta.-subunit of RNA polymerase is useful as a criterion for mycobacterial phylogenetic analysis, based on the following reports:
RNA polymerase gene, besides the said criteria for phylogenetic analysis, can be used as an alternative of 16S rRNA gene, since RNA polymerase has subunits of rpoA, rpoB, rpoc and rpoD which are highly conserved throughout procaryotes (see: Lill, U. I., E. M. Behrendt and G. R. Hartmann, Eur. J. Biochem., 52:411-420(1975)).
Among the subunits, the rpoB gene coding for .beta.-subunit of RNA polymerase is related to rifampin resistance in Escherichia coli (see: Jin, D. and C. A. Gross, Mapping and Sequencing of Mutations in the Escherichia coli rpoB Gene that Lead to Rifampicin Resistance, J. Mol. Biol., 202:45-58(1988)), Mycobacterium tuberculosis (see: Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer and T. Bodmer, Detection of Rifampin-resistance Mutations in Mycobacterium tuberculosis, Lancet, 341:647-650(1993)), Mycobacterium leprae (see: Honore, N. T., Bergh, S., Chanteau, S., Doucet-Populaire, F., Eiglmeier, K., Garnier, T., Georges, C., Launois, P., Limpaiboon, T., Newton, S., Niang, K., Del Portillo, P., Ramesh, G. R., Reddi, P., Ridel, P. R., Sittisombut, N., Wu-Hunter, S. and Cole, S. T., Nucleotide Sequence of the First Cosmid from the Mycobacterium leprae Genome Project: Structure and Function of the Rif-Str Regions, Mol. Microbiol., 7(2):207-214(1993)) and M. smegmatis (see: Levin, M. E. and Hatfull, G. F., Mycobacterium smegmatis RNA Polymerase: DNA Supercoiling, Action of Rifampin and Mechanism of Rifampin Resistance, Mol. Microbiol., 8(2):277-285(1993)).
Also, nucleotide sequence in a region of a rpoB gene is highly conserved in some mycobacteria other than Mycobacterium tuberculosis (see: Hunt, J. M., G. D. Roberts, L. Stockman, T. A. Felmiee and D. H. Persing, Detection of a Genetic Locus Encoding Resistance to Rifampin in Mycobacterial Cultures and in Clinical Specimens, Diagn. Microbiol. Infect. Dis., 18:219-272(1994); Whelen, A. C., T. A. Felmlee, J. M. Hunt, D. L. Williams, G. D. Roberts, L. Stockman and D. H. Persing, Direct Genotypic Detection of Mycobacterium tuberculosis Rifampin Resistance in Clinical Specimens by Using Single-tube Heminested PCR, J. Clin. Microbiol., 33:556-561(1995)).
In addition, a rpoB gene is used for phylogenetic establishment of Archaebacteria(see: Puhler, G., H. Leffers, F. Gropp, P. Palm, H. P. Klenk, F. Lottspeich, R. A. Garrett and W. Zillig, Archaebacterial DNA-dependent RNA Polymerases Testify to the Evolution of the Eukaryotic Nuclear Genome, Proc. Natl. Acad. Sci., U.S.A., 86:4569-4573(1989); Iwabe N., K. Kuma, H. Kishino, M. Hasegawa, and Miyata, Evolution of RNA Polymerases and Branching Patterns of the Three Major Groups of Archaebacteria, J. Mol. Evol., 32:70-78(1991); Klenk, H. P. and W. Zillg, DNA-dependent RNA Polymerase Subunit B as a Tool for Phylogenetic Reconstructions: Branching Topology of the Archaeal Domain, J. Mol. Evol., 38:420-432(1994); Zillig, W., H. P. Klenk, P. Palm, G. Puhler, F. Gropp, R. A. Garrett and H. Leffers, The Phylogenetic Relations of DNA-dependent RNA Polymerases of Archaebacteria, Eukaryotes, and Eubacteria, Can. J. Microbiol., 35:73-80(1989)), Eubacteria other than Staphylococcus aureus (see: Rowland G. C., M. Aboshkiwa and G. Coleman, Comparative Sequence Analysis and Predicted Phylogeny of the DNA-dependent RNA Polymerase Beta Subunits of Staphylococcus aureus and other Eubacteria, Biochem. Soc. Trans., 21:40S(1993)) and Plasmodium (see: Gardner, M. J., N. Goldman, P. Barnett, P. W. Moore, K. Rangachari, M. Strath, A. Whyte, D. H. Williamson and R. J. Wilson, Phylogenetic Analysis of the rpoB Gene from the Plastid-like DNA of Plasmodium falciparum, Mol. Biochem. Parasitor., 66:221-231(1994)).